Skin repair composition as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of cosmetics, and particularly relates to a skin repairing composition as well as a preparation method and application thereof. The skin repair composition consists of a premix and a bletilla striata water extract, the premix is prepared from naringenin chalcone, apigenin, L-glutamic acid, 2-aminoisobutyric acid, procyanidine and caffeic acid according to the mass ratio of (0.001 to 0.005) to (0.01 to 0.05) to (0.01 to 0.05) to (0.02 to 0.06) to (0.01 to 0.03) to (0.04 to 0.08); the mass ratio of the premix to the water extract of the bletilla striata flowers is (0.1-0.3): (0.0075-0.15). The in-vitro antioxidant activity of the obtained skin repair composition is remarkably improved, the intracellular ROS level is effectively reduced, oxidative stress injury is relieved, secretion of inflammatory factors in cells can be remarkably reduced, the cells are protected against inflammatory injury caused by UVB, and the skin repair composition further has remarkable anti-aging and moisturizing activity.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cosmetics, and particularly relates to a skin repair composition and a preparation method and application thereof. BACKGROUND

[0002] Ultraviolet radiation is one of the common factors affecting skin health, and the skin inflammation induced by medium wave ultraviolet (UVB) mainly manifests as up-regulation of reactive oxygen species (ROS) level, migration and chemotaxis of inflammatory cells, and expression and release of inflammatory factors.

[0003] Bletilla striata, widely planted in Yunnan, Guizhou, Hunan and other regions, is a traditional medicinal and edible plant, which can be used for clinical medicine, industry and beauty, etc., and has a wide range of applications. As the flower of Bletilla striata of Orchidaceae, Bletilla striata flower has rich color and brightness, and is rich in active substances such as anthocyanins, polyphenols and flavonoids. Except for a small amount of Bletilla striata flower being used for ornamental, most of the Bletilla striata flower is discarded, which causes great waste of Bletilla striata resources. It is found that the active substances such as anthocyanins, polyphenols, flavonoids and polysaccharides in Bletilla striata are related to the antioxidant, anti-inflammatory, moisturizing and anti-aging activities thereof, but the antioxidant, anti-inflammatory, moisturizing and anti-aging activities of Bletilla striata extract are weak. SUMMARY

[0004] In order to solve the above technical problems, the present application provides a skin repair composition and a preparation method and application thereof.

[0005] A skin repair composition, which is composed of a premix and a Bletilla striata flower water extract; The premix is composed of naringenin chalcone, apigenin, L-glutamic acid, 2-amino isobutyric acid, proanthocyanidin and caffeic acid in a mass ratio of 0.001-0.005:0.01-0.05:0.01-0.05:0.02-0.06:0.01-0.03:0.04-0.08. The mass ratio of the premix to the Bletilla striata flower water extract is 0.1-0.3:0.0075-0.15.

[0006] Preferably, the mixture is composed of naringenin chalcone, apigenin, L-glutamic acid, 2-amino isobutyric acid, proanthocyanidin and caffeic acid in a mass ratio of 0.002:0.03:0.03:0.04:0.02:0.06.

[0007] Preferably, the mass ratio of the premix to the Bletilla striata flower water extract is 0.182:0.09.

[0008] Preferably, the preparation method of the Bletilla striata flower water extract is as follows: Bletilla striata flower powder and water are mixed in a ratio of 1g:20mL-50mL, and then extracted at 60℃-90℃ for 1h-3h by refluxing, and then the filtrate is concentrated and freeze-dried to obtain the Bletilla striata flower water extract.

[0009] A preparation method of the skin repair composition, comprising the following steps: The naringenin chalcone, apigenin, L-glutamic acid, 2-amino isobutyric acid, proanthocyanidin and caffeic acid are mixed according to the mass ratio to obtain the premix; The premix is uniformly mixed with the Bletilla striata flower water extract to obtain the skin repair composition.

[0010] Preferably, the preparation method of the Bletilla striata flower water extract is that the Bletilla striata flower powder and water are mixed according to the ratio of 1g:20mL-50mL, and are extracted at 60-90℃ under reflux for 1-3h, and the filtrate is concentrated and freeze-dried to obtain the Bletilla striata flower water extract.

[0011] The skin repair composition is applied to the preparation of a skin repair product.

[0012] Preferably, the skin repair product further comprises one or more than two combinations of moisturizing agents, thickening agents, emulsifying agents, chelating agents, preservatives and solvents.

[0013] Preferably, the solvent is water. The skin repair product is a solution with a concentration of 0.125mg / mL-2mg / mL prepared by diluting the skin repair composition with water.

[0014] Preferably, the repair is at least one of antioxidant, anti-aging, UV damage repair and moisturizing.

[0015] Compared with the prior art, the skin repair composition has the beneficial effects that: The skin repair composition prepared by mixing naringenin chalcone, apigenin, L-glutamic acid, 2-amino isobutyric acid, proanthocyanidin and caffeic acid according to the mass ratio of 0.001-0.005:0.01-0.05:0.01-0.05:0.02-0.06:0.01-0.03:0.04-0.08 and then mixing with the Bletilla striata flower water extract has a significantly increased in-vitro antioxidant activity, effectively reduces the intracellular ROS level, reduces oxidative stress damage, and can significantly reduce the secretion of inflammatory factors in cells, protects cells from inflammatory damage caused by UVB, and has significant anti-aging and moisturizing activity.

[0016] Among them, the polysaccharides in the water extract of Bletilla striata flower can directly scavenge free radicals and improve the activities of antioxidant enzymes such as SOD and CAT, and the phenanthrene / bibenzyl compounds can reduce the generation of ROS, and the procyanidins, caffeic acid, naringenin chalcone and apigenin as flavonoids / polyphenols can directly neutralize ROS. The enzyme activation of Bletilla striata polysaccharide and the synergistic effect of flavonoids / polyphenols significantly reduce the ROS level induced by UVB; caffeic acid and procyanidins form antioxidant complexes with Bletilla striata polysaccharides through hydrogen bonding / hydrophobic interaction, and the stability is enhanced; apigenin and glutamic acid form flavonoid-amino acid adducts through amidation reaction, which improves cell permeability; phenanthrene compounds and naringenin chalcone in the water extract of Bletilla striata flower are co-localized in the cell membrane through hydrophobic interaction, and synergistically scavenge lipid free radicals, forming an antioxidant-anti-inflammatory-anti-aging-hydrating synergistic relationship.

[0017] The anthocyanins in the water extract of Bletilla striata flower reduce collagen degradation, the polysaccharides promote fibroblast proliferation, increase collagen secretion, and the procyanidins stimulate collagen synthesis, and the 2-amino isobutyric acid enhances the stability of collagen, and together with the Bletilla striata polysaccharide, they resist aging; the Bletilla striata polysaccharide forms a polysaccharide-protein complex, enhances the water-locking ability of the stratum corneum, and L-glutamic acid as a natural moisturizing factor (NMF) component maintains skin hydration, and the physical barrier of Bletilla striata polysaccharide and the chemical hygroscopic effect of glutamic acid synergistically provide long-lasting hydration; at the same time, the synthesis of collagen is enhanced, which in turn promotes the increase of collagen fiber density by procyanidins, apigenin and caffeic acid, so that the dermal structure is more compact, reducing water evaporation, and the Bletilla striata polysaccharide and 2-amino isobutyric acid increase the content of endogenous moisturizing factors by increasing the content of endogenous moisturizing factors, forming a mutually synergistic effect.

[0018] In the synergistic effect formed, 2-amino isobutyric acid itself does not have free radical scavenging ability and total reducing power, but after being added to the composition, it improves the antioxidant efficacy of Bletilla striata, and further promotes the formation of an antioxidant-anti-inflammatory-anti-aging-hydrating synergistic relationship. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The in vitro antioxidant activity of Bletilla striata flower water extract and the composition was determined, wherein A is the ABTS free radical scavenging rate, B is the DPPH free radical scavenging rate, and C is the total reducing power.

[0020] Figure 2 The CCK-8 was used to detect the cell viability after incubation for 48 hours in each group.

[0021] Figure 3 The ROS content accumulation change.

[0022] Figure 4 The protective effect of the composition and the water extract of Bletilla striata flower on the skin inflammatory reaction caused by UVB, wherein A is the expression of IL-6, and B is the expression of TNF-α.

[0023] Figure 5 The moisture absorption and retention capacity of the composition, the water extract of Bletilla striata and glycerin in the environment of relative humidity 43% and 83%, wherein A is the moisture absorption capacity at relative humidity 43%, B is the moisture absorption capacity at relative humidity 83%, C is the moisture retention capacity at relative humidity 43%, and D is the moisture retention capacity at relative humidity 83%.

[0024] Figure 6 The longevity activity of the composition and the water extract of Bletilla striata.

[0025] Figure 7 The health activity of the composition and the water extract of Bletilla striata, A: the number of pharyngeal pumps, B: the number of body swings, C: the reproductive capacity.

[0026] Figure 8 The stress resistance activity of the composition and the water extract of Bletilla striata, A: heat stress, B: oxidative stress, C: ultraviolet stress. DETAILED DESCRIPTION

[0027] The specific embodiments of the present application are described in detail below, but it should be understood that the scope of protection of the present application is not limited by the specific embodiments. Based on the examples in the present application, all other examples obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present application. The experimental methods described in the embodiments of the present application are conventional methods unless otherwise specified.

[0028] Example 1 A skin care composition, consisting of a premix and a water extract of Bletilla striata.

[0029] The premix consists of naringenin chalcone, apigenin, L-glutamic acid, 2-amino isobutyric acid, proanthocyanidin, and caffeic acid in a mass ratio of 0.002g:0.03g:0.03g:0.04g:0.02g:0.06g.

[0030] The mass ratio of the premix to the water extract of Bletilla striata is 0.182g:0.09g.

[0031] Example 2 A skin care composition, consisting of a premix and a water extract of Bletilla striata.

[0032] The premix consists of naringenin chalcone, apigenin, L-glutamic acid, 2-amino isobutyric acid, proanthocyanidin, and caffeic acid in a mass ratio of 0.001g:0.05g:0.05g:0.02g:0.01g:0.08g.

[0033] The mass ratio of the premix to the water extract of Bletilla striata is 0.211g:0.06g.

[0034] Example 3 A skin care composition, consisting of a premix and a Bletilla striata flower water extract.

[0035] The premix consists of naringenin chalcone, apigenin, L-glutamic acid, 2-amino isobutyric acid, proanthocyanidin, caffeic acid in a mass ratio of 0.005g:0.01g:0.01g:0.06g:0.03g:0.04g.

[0036] The mass ratio of the premix to the Bletilla striata flower water extract is 0.155g:0.15g.

[0037] The skin care compositions in Examples 1-3 are configured into composition solutions of corresponding concentrations in subsequent experiments, and the composition solution configuration method is as follows: the Bletilla striata flower water extract is configured into a Bletilla striata flower water extract solution with distilled water, and the premix is added to the Bletilla striata flower water extract solution to obtain the composition solution.

[0038] Example 4 A preparation method of the skin care composition in Example 1: 0.002g naringenin chalcone, 0.03g apigenin, 0.03g L-glutamic acid, 0.04g 2-amino isobutyric acid, 0.02g proanthocyanidin, and 0.06g caffeic acid are weighed according to the proportions of the raw materials in Example 1, mixed, and uniformly mixed with 0.09g Bletilla striata flower water extract to obtain the skin care composition.

[0039] The preparation methods of the skin care compositions of Examples 2 and 3 are the same as the preparation method of the skin care composition of Example 1.

[0040] Example 5 A preparation method of the Bletilla striata flower water extract: 1:30 g / mL of Bletilla striata powder is added to distilled water, and extracted twice at 90°C for 1h, the filtrates are combined, and rotary evaporation is performed to 1 / 3 of the volume, and then freeze-dried at -80°C to obtain a freeze-dried powder, which is the Bletilla striata flower water extract.

[0041] In subsequent experiments, the freeze-dried powder is configured into a Bletilla striata flower water extract solution of a corresponding concentration with distilled water.

[0042] Example 6 A preparation method of the skin care composition in Example 1: 0.002g naringenin chalcone, 0.03g apigenin, 0.03g L-glutamic acid, 0.04g 2-amino isobutyric acid, 0.02g proanthocyanidin, and 0.06g caffeic acid are weighed according to the proportions of the raw materials in Example 1, mixed, and uniformly mixed with 0.09g Bletilla striata flower water extract to obtain the skin care composition.

[0043] Preparation method of the water extract of Bletilla striata flower: 1:30 g / mL of Bletilla striata flower powder was added to distilled water, and refluxed at 90°C for 1 h twice. The combined filtrate was rotary evaporated to 1 / 3 of the volume, and then freeze-dried at -80°C to obtain a freeze-dried powder, which was the water extract of Bletilla striata flower.

[0044] Comparative Example 1 A composition consisting of a premix and the water extract of Bletilla striata flower.

[0045] The premix consists of naringenin chalcone, apigenin, L-glutamic acid, proanthocyanidin, and caffeic acid in a mass ratio of 0.002 g:0.03 g:0.03 g:0.02 g:0.06 g.

[0046] The mass ratio of the premix to the water extract of Bletilla striata flower is 0.142 g:0.09 g.

[0047] Effect verification I. The skin care composition is denoted as HF, and the water extract of Bletilla striata flower is denoted as BF, and the following verification is performed.

[0048] 1. Evaluation of the in vitro antioxidant activity of the composition and the water extract of Bletilla striata flower ① DPPH free radical scavenging experiment: 2 mL of 0.1 mmol / L DPPH ethanol solution was added to 2 mL of water extract solution with concentrations of 0.0625 mg / mL, 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 0.75 mg / mL, 1 mg / mL, 1.5 mg / mL, and 2 mg / mL, respectively, to obtain a mixed solution. The mixed solution was reacted in the dark for 30 min, and the absorbance A1 of the water extract solution was measured at 517 nm. 2 mL of water extract solution and 2 mL of anhydrous ethanol were used as a blank, and the absorbance A2 of the water extract solution was measured at 517 nm. 2 mL of anhydrous ethanol and 2 mL of DPPH solution were used as a control, and the absorbance A0 was measured at 517 nm.

[0049] 2 mL of 0.1 mmol / L DPPH ethanol solution was added to 2 mL of the composition solution with different concentrations respectively to obtain a mixed solution, the mixed solution was reacted for 30 min in the dark, and the absorbance A1 of the composition solution was measured at 517 nm; 2 mL of the composition solution and 2 mL of anhydrous ethanol were used as a blank, and the absorbance A2 of the composition solution was measured at 517 nm; 2 mL of anhydrous ethanol and 2 mL of DPPH solution were used as a control, and the absorbance A0 was measured at 517 nm. The concentrations of the water extract in the composition solution with different concentrations were 0.0625 mg / mL, 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 0.75 mg / mL, 1 mg / mL, 1.5 mg / mL and 2 mg / mL respectively, and the amounts of other raw materials remained unchanged.

[0050] The 0.1 mmol / L DPPH ethanol solution was prepared by dissolving 0.002 g of DPPH in 50 mL of anhydrous ethanol.

[0051] VC solution was used as a positive control, and the DPPH free radical scavenging activity of the composition and the water extract of Bletilla striata flower was calculated according to the following formula: DPPH scavenging rate % = [1 - (A1-A2) / A0] x 100% ABTS free radical scavenging experiment: 0.5 mL of the water extract of Bletilla striata flower solution with concentrations of 0.0625, 0.125, 0.25, 0.5, 0.75, 1, 1.5 and 2 mg / mL respectively was taken in a test tube, 3 mL of ABTS free radical solution was added to obtain a mixed solution, the mixed solution was reacted for 5 min in the dark at room temperature, and the absorbance A1 of the water extract of Bletilla striata flower solution was measured at 734 nm; the above operation was repeated by replacing the water extract of Bletilla striata flower solution with distilled water, and the absorbance A0 of the water extract of Bletilla striata flower solution was measured at 734 nm; the above operation was repeated by replacing the ABTS free radical solution with distilled water, and the absorbance A2 of the water extract of Bletilla striata flower solution was measured at 734 nm; 0.5 mL of the composition solution with different concentrations was taken in a test tube, 3 mL of ABTS free radical solution was added to obtain a mixed solution, the mixed solution was reacted for 5 min in the dark at room temperature, and the absorbance A1 of the composition solution was measured at 734 nm; the above operation was repeated by replacing the composition solution with distilled water, and the absorbance A0 of the composition solution was measured at 734 nm; the above operation was repeated by replacing the ABTS free radical solution with distilled water, and the absorbance A2 of the composition solution was measured at 734 nm; the concentrations of the water extract in the composition solution with different concentrations were 0.0625 mg / mL, 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 0.75 mg / mL, 1 mg / mL, 1.5 mg / mL and 2 mg / mL respectively, and the amounts of other raw materials remained unchanged.

[0052] ABTS radical solution was prepared by mixing 7 mmol / L ABTS solution with 2.45 mmol / L K2S2O8 solution at a ratio of 1:1, and then reacting in dark for 12 hours at room temperature. The ABTS radical solution was obtained by diluting the mother liquor with distilled water to an absorbance value of 0.7±0.02.

[0053] The ABTS radical scavenging rate of the composition and the Bletilla striata flower extract was calculated according to the following formula, with VC solution as the positive control: ABTS radical scavenging rate (%) = [1-(A1-A2) / A0]×100% ③Total reducing capacity experiment: 2 mL of the Bletilla striata flower extract solution with different concentrations and 2 mL of the composition solution with different concentrations were added into a 10 mL test tube, followed by sequentially adding 2 mL of phosphate buffer (0.2 mol / L, pH=6.6) and 2 mL of 1% potassium ferricyanide, and then reacting in a 50℃ water bath for 20 min. After cooling, 2 mL of 10% trichloroacetic acid was added, and then mixed and allowed to stand. After that, 2 mL of distilled water and 0.5 mL of 0.1% ferric trichloride solution were added, and then mixed and allowed to stand for 15 min. The absorbance of the Bletilla striata flower extract solution and the composition solution was measured at 700 nm with distilled water as the blank reference liquid. VC was used as the positive control, and A 700 nm was greater, the stronger the reducing capacity was. The concentrations of the Bletilla striata flower extract solution with different concentrations were 0.0625 mg / mL, 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 0.75 mg / mL, 1 mg / mL, 1.5 mg / mL and 2 mg / mL, respectively. The concentrations of the Bletilla striata flower extract solution in the composition solution with different concentrations were 0.0625 mg / mL, 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 0.75 mg / mL, 1 mg / mL, 1.5 mg / mL and 2 mg / mL, respectively. The dosages of other raw materials were unchanged.

[0054] 2. UV damage repair and anti-inflammatory activity of the composition and the Bletilla striata flower extract ①Evaluation of the toxicity of honey-baked Bletilla striata flower and Bletilla striata flower on HaCaT cells When HaCaT cells (human immortalized epidermal keratinocytes, purchased from Wuhan Pronosei Biotechnology Co., Ltd.) reached 80% confluence, 100 μL of cell suspension was digested and seeded into 96-well plates. After cell attachment, 1.5 mg / mL of the combined solution and 1.5 mg / mL of Bletilla striata flower water extract solution were added to the 96-well plates and incubated for 24 h. After incubation, 10 μL of CCK8 solution (purchased from MedChemexpress Biotechnology Co., Ltd.) was added to each well, and the plates were incubated at 37°C in the dark for 40 min. The absorbance of each well was measured at 450 nm using a microplate reader.

[0055] ② Effects of the composition and Bletilla striata flower water extract on ROS clearance in HaCaT cells under UV irradiation HaCaT cell suspension was seeded into 96-well plates. After cell adhesion, the cells were treated with 1.5 mg / mL of the combined solution, 1.5 mg / mL of Bletilla striata flower water extract solution, and 5 μg / mL of the positive control VC solution for 8 h, respectively. Subsequently, the cell culture plates were placed under a UV crosslinker to construct a UV damage model at 750 mJ / cm². 2 Irradiation was performed for 30 minutes under the specified conditions. After irradiation, the ROS levels in each group of cells were measured using a ROS detection kit (purchased from Beijing Solarbio Science & Technology Co., Ltd.).

[0056] ③ Effects of the composition and Bletilla striata flower water extract on inflammation of HaCaT cells under ultraviolet irradiation Measurement of cytokine IL-6 and TNF-α secretion levels: HaCaT cells were administered at a rate of 5 × 10⁻⁶ cells / year. 6 The concentration was seeded in 10cm cell culture dishes. 750mJ / cm 2 After UVB irradiation, a 1.5 mg / mL solution of the combined composition, a 1.5 mg / mL solution of Bletilla striata flower water extract, and a 5 μg / mL solution of Vitamin C were prepared in DMEM basal medium and incubated for another 6 hours. The IL-6 level was then measured using IL-6 ELISA and TNF-α detection kits.

[0057] 3. Moisturizing activity of the composition and Bletilla striata flower water extract At 20℃, using glycerol as a control, the moisture absorption rates of the HF and BF groups were determined in two sealed environments (43% relative humidity for saturated sodium carbonate solution and 83% relative humidity for saturated potassium chloride solution). After drying the samples at 60℃ for 24 hours, the mass was recorded as M1 (g). The samples were then placed in sealed containers under constant temperature and humidity, and weighed at 2h, 4h, 6h, 8h, 10h, and 12h, and recorded as M2 (g). The moisture absorption rate was calculated as a percentage (%): Moisture absorption rate = (M2 / M1) × 100.

[0058] The control glycerol, HF and BF were prepared into 500 μg / mL solution with deionized water, and the moisture retention rate of the sample solution in two closed environments (relative humidity of saturated sodium carbonate solution 43%, relative humidity of saturated potassium chloride solution 83%) was determined. Before testing, the sample solution was weighed and recorded as M3, g, and placed in a closed container with constant temperature and humidity. After 2h, 4h, 6h, 8h, 10h and 12h, the weight was recorded as M4, g. The moisture retention rate was calculated, and the unit was %. Moisture retention rate = (M4 / M3) x 100.

[0059] 4. Anti-aging activity of the composition and the white flower water extract ① Longevity activity experiment: synchronized L4 stage nematodes were picked onto the blank group, HF group and BF group NGM medium (containing FUDR) with added E. coli food in advance, recorded as day 0, 30 nematodes per dish, and the medium was replaced every two days. The nematodes grew on the drug-containing medium for the first ten days, and then were transferred to the medium without drugs. The number of surviving nematodes was counted every day, and the dead nematodes (nematodes that did not move and did not respond after being stimulated by platinum wire for 10 seconds were considered dead) were picked out of the culture dish, and the nematodes with external genitalia rupture and wall-drying were excluded from the sample, until all the nematodes died.

[0060] ② Pharyngeal pump, body swing and reproduction experiment Pharyngeal pump: the nematodes in the blank group, HF group and BF group were raised according to the nematode longevity experiment method, and the pharyngeal pump swallowing times of each nematode (15-20 nematodes per group) in 30s were tested every other day starting from the second day, and observed on the 13th day. Prepare blank medium without E. coli, and randomly pick one nematode in good condition to be tested and place it on the blank medium. After waiting for a period of time until the overreaction of the nematode to external stimuli is alleviated, observe and record the number of pharyngeal pumps in 30s under a stereomicroscope.

[0061] Body swing: the number of body swings of the nematodes in 30s was recorded according to the same method. Once the nematode bends to the left or right side of the body, it is considered as one swing.

[0062] Nematode reproduction: after synchronization, the nematodes in L4 stage were picked into NGM dishes containing the corresponding concentration of drugs, 1-3 nematodes per dish, 3 dishes per group, and transferred to new NGM dishes every 24h for 5-6 days until ovulation was completed. The number of offspring was counted after the egg-containing dishes were cultured for 48h. The number of offspring of each dish was added to obtain the total number of offspring of the nematodes.

[0063] ③ Stress experiment Heat stress experiment: After nematode synchronization treatment, nematodes cultured to the L4 stage were transferred to NGM culture dishes containing the corresponding drug concentrations. The drug groups were 1 mg / mL, 1.5 mg / mL, and 2 mg / mL. A blank group was set up without drug. Each dish contained 20-30 nematodes, with 3 dishes per group. The dishes were cultured in a constant temperature incubator at 20℃ for 5 days. 20 nematodes were then transferred to blank culture dishes (which needed to be preheated in a 35℃ incubator for 30 min) and placed in a 35℃ incubator for heat stress. The loss and survival of nematodes were observed and recorded every 1-2 hours. The observation was continued for 10 hours or until no nematodes survived in the dish. The criteria for nematode death were: no movement, no swallowing, and no response to touch by the fine platinum wire.

[0064] Oxidative stress experiment: After nematode synchronization treatment, nematodes cultured to the L4 stage were selected and placed into NGM culture dishes containing the corresponding concentration of drug, 20-30 nematodes per dish, 3 dishes per group, and cultured in a constant temperature incubator at 20℃ for 5 days. Then, 20 nematodes were transferred to blank culture dishes containing 30mmol / L H2O2 (0.154mL of 30% H2O2 was taken and diluted to 50mL). The loss and survival of nematodes were observed and recorded every 0.5h-1h until all nematodes died. The criteria for judging the death of nematodes were: no movement, no swallowing, and no response when touched by fine platinum wire.

[0065] UV stress experiment: After nematode synchronization treatment, nematodes cultured to the L4 stage were selected and placed in NGM culture dishes containing the corresponding concentration of drug, 20 to 30 nematodes per dish, 3 dishes per group, and cultured in a constant temperature incubator at 20℃ for 5 days. Then, 20 nematodes were transferred to a culture medium without E. coli, the lid was opened, and the nematodes were placed under direct UV irradiation. The number of dead and surviving nematodes was counted every 0.5 to 1 hour until all of them died.

[0066] result I. Comparison of results between skin repair composition HF and Bletilla striata flower water extract BF 1. Evaluation of the in vitro antioxidant activity of the composition and Bletilla striata flower water extract Changes in the in vitro antioxidant activity of the composition compared to that of Bletilla striata flower extract alone are as follows: Figure 1 Compared with the water extract of Bletilla striata flower alone, the composition exhibits stronger free radical scavenging ability and total reducing power. The results indicate that the composition can synergistically enhance the antioxidant efficacy of Bletilla striata flower.

[0067] 2. UV damage repair and anti-inflammatory activities of the composition and Bletilla striata flower water extract like Figure 2 As shown, the combination and the water extract of Bletilla striata flower had no effect on the viability of HaCaT cells, indicating that Bletilla striata flower has no cytotoxicity and good safety.

[0068] like Figure 3As shown, compared with the control group, the content of ROS in HaCaT cells after UVB-induced damage increased significantly. After the composition and Bletilla striata flower water extract protected the cells, the cells were irradiated with UVB, and the content of ROS was significantly lower than that of the model group. The protective effect of the composition on the oxidative stress response in HaCaT cells caused by UVB irradiation was better than that of Bletilla striata flower water extract alone.

[0069] According to the experimental results in Figure 4 It can be seen that after UVB irradiation, the content of IL-6 and TNF-α pro-inflammatory factors in the model group increased significantly, causing cell inflammatory infiltration. The content of pro-inflammatory factors in the sample group decreased significantly, and the inhibitory effect of the composition on cell inflammation was better than that of Bletilla striata flower water extract alone. The results showed that the composition could effectively protect the cells from damage caused by UVB and relieve cell inflammation.

[0070] 3. Moisturizing activity of the composition and Bletilla striata flower water extract Figure 5 A shows that the moisture absorption rates of the composition and Bletilla striata flower water extract are not much different, and are higher than that of the control glycerol in an environment of 20°C and 43% relative humidity.

[0071] Figure 5 B shows that the moisture absorption rates of the composition and Bletilla striata flower water extract are both >145.50%, slightly lower than that of the control glycerol (152.28%) in an environment of 20°C and 83% relative humidity. In summary, the composition has stronger moisture absorption in both relative humidity environments.

[0072] Figure 5 C shows that in an environment of 20°C and 43% relative humidity, the moisture retention rates of all samples are >86.68% at 24h, showing good moisturizing effect. Among them, the moisture retention rates of the composition and Bletilla striata flower water extract are not much different from that of the control glycerol, and the moisture retention rate of the composition is higher than that of Bletilla striata flower water extract alone.

[0073] Figure 5 D shows that in an environment of 20°C and 83% relative humidity, the moisture retention rates of all samples remain above 82.35% at 24h, showing excellent moisturizing performance. Among them, the moisture retention rates of the composition and Bletilla striata flower water extract are better than that of the control glycerol.

[0074] 4. Anti-aging activity of the composition and Bletilla striata flower water extract The longevity experiment results of C. elegans show that the median survival time, average lifespan, and maximum lifespan of the composition and the B. officinalis flower water extract are higher than those of the blank control group, indicating that both have certain longevity activity. And the composition group achieves the best longevity activity, with a median lifespan of 21 days, an average lifespan of 22.68 days, and a maximum lifespan of 33 days. Compared with the blank group, the average lifespan is increased by 30%. In the B. officinalis flower water extract group alone, the longevity of C. elegans is prolonged, and the median lifespan is increased to 20 days, the average lifespan is increased to 21.2 days, and the maximum lifespan is increased to 32 days, and the average lifespan is increased by 22.23%. The composition and the B. officinalis flower water extract both show significant longevity activity, and the average lifespan of the composition group is increased by 10.54% compared with the B. officinalis flower water extract group alone. Figure 6 .

[0075] With the increase of age, the pharyngeal pumping rate of C. elegans gradually decreases. Notably, the reduction of food intake leads to the heat restriction effect, and the occurrence of heat restriction leads to the prolongation of the lifespan of adult animals in many animals. Therefore, the pharyngeal pumping frequency of the nematode is also an important factor for investigating the function of the nematode body, and the pharyngeal pumping frequency reflects the feeding capacity of the nematode. The experimental results of the present application show that the pharyngeal pumping frequency of the nematode decreases with the increase of days, and the B. officinalis flower treatment group shows a trend of improving the pharyngeal pumping frequency of the nematode from the 5th day Figure 7 A).

[0076] The body swinging frequency of C. elegans is also one of the indicators for measuring the aging of the nematode. The movement of the nematode usually presents a sinusoidal curve, and the influence of the drug on the movement ability of the nematode can be evaluated by the body swinging frequency of the nematode. The experimental results of the present application show that the body swinging frequency of the nematode decreases with the increase of days, and the B. officinalis flower treatment group shows a trend of improving the body swinging frequency of the nematode to different degrees from the 3rd day, and has a significant difference in the 7th-13th day (*P<0.05) Figure 7 B).

[0077] By counting the total number of offspring of the nematodes treated with the blank group, the composition and the B. officinalis flower water extract, whether the composition and the B. officinalis flower water extract can affect the reproductive capacity of the nematode is observed. The experiment shows that the composition can significantly increase the total egg production, and the B. officinalis flower water extract can increase the egg production, but there is no significant difference Figure 7 C).

[0078] The lifespan of the body is closely related to the tolerance of the anti-stress ability, and in the case of external pressure, the stronger the tolerance of the anti-stress ability, the longer the lifespan. After giving the nematode high-temperature stimulation, observing the change of the lifespan of the nematode is helpful to analyze whether the lifespan of the nematode is related to the tolerance of the anti-heat stress ability. In the high-temperature stimulation at 35℃, the average lifespan of the composition group is obviously increased compared with the blank group, and it is considered that the longevity of the composition is related to the tolerance of the anti-heat stress ability.Figure 8 A).

[0079] Similarly, the change of the life span of the nematode after being given H2O2 and ultraviolet stimulation is helpful to analyze whether the life span of the nematode is related to the antioxidant and ultraviolet stress tolerance of the nematode. The average life span of the composition under the stimulation of 30 mmol / L H2O2 and ultraviolet is obviously increased compared with the blank group. Figure 8 B, C).

[0080] The present application takes Bletilla striata flower as the research object, and studies the protection of skin inflammation reaction and barrier damage caused by UVB irradiation, antioxidant activity, anti-aging, moisturizing and other activities of the water extract alone and the combination of the water extract and the compound. The research results show that the in vitro antioxidant activity of the combination of Bletilla striata flower is significantly increased. Through the cell model induced by UVB damage, the composition can effectively reduce the intracellular ROS level, alleviate oxidative stress damage, and significantly reduce the secretion of inflammatory factors in cells after UVB stimulation, and protect cells from inflammatory damage caused by UVB. The composition also has significant anti-aging activity.

[0081] II. Comparison of D1 and 2-aminoisobutyric acid with skin care composition HF.

[0082] The present application also studies the results of the composition of Comparative Example 1 and 2-aminoisobutyric acid in the above experiments. The composition of Comparative Example 1 is denoted as D1.

[0083] 1. Evaluation of in vitro antioxidant activity The free radical scavenging capacity and total reducing capacity of D1 and 2-aminoisobutyric acid at a concentration of 1.5 mg / mL were detected, and the concentration count and detection method were the same as above.

[0084] The free radical scavenging capacity and total reducing capacity of each group are shown in Table 1. As can be seen from the results, 2-aminoisobutyric acid does not have free radical scavenging capacity and total reducing capacity, and the free radical scavenging capacity and total reducing capacity of D1 are better than those of BF. The results show that 2-aminoisobutyric acid can synergistically improve the antioxidant efficacy of Bletilla striata flower.

[0085] Table 1. Free radical scavenging capacity and total reducing capacity of each group It should be noted that when numerical ranges are involved in the claims of the present application, both endpoints of each numerical range and any number between the two endpoints can be selected, and in order to prevent repetition, the present application describes preferred embodiments.

[0086] While the preferred embodiments of the application have been described, additional variations and modifications can be made to these embodiments by those skilled in the art once they have the benefit of the present disclosure without departing from the spirit and scope of the application. Accordingly, it is intended that the appended claims include all such modifications and variations as fall within the scope of the present application.

[0087] It is apparent that those skilled in the art can make various changes and modifications to the application without departing from the spirit and scope of the application. It is therefore intended that the present application cover all such changes and modifications that are within its scope.

Claims

1. A skin care composition characterized in that, consists of a premix and a Bletilla striata flower water extract; the premix consists of naringin chalcone, apigenin, L-glutamic acid, 2-amino isobutyric acid, proanthocyanidin, caffeic acid in a mass ratio of 0.001-0.005:0.01-0.05:0.01-0.05:0.02-0.06:0.01-0.03:0.04-0.08; the mass ratio of the premix to the Bletilla striata flower water extract is 0.1-0.3:0.0075-0.

15.

2. The skin treatment composition of claim 1, wherein, the mixture consists of naringin chalcone, apigenin, L-glutamic acid, 2-amino isobutyric acid, proanthocyanidin, caffeic acid in a mass ratio of 0.002:0.03:0.03:0.04:0.02:0.

06.

3. The skin treatment composition of claim 1, wherein the mass ratio of the premix to the Bletilla striata flower water extract is 0.182:0.

09.

4. The skin treatment composition of claim 1, wherein, The preparation method of the Bletilla striata flower water extract is as follows: Bletilla striata flower powder and water are mixed in a ratio of 1g:20mL-50mL, and extraction is carried out at 60-90℃ under reflux for 1-3h, and the filtrate is concentrated and freeze-dried to obtain the Bletilla striata flower water extract.

5. A method of preparing the skin care composition of claim 1, characterized in that, comprises the following steps: naringin chalcone, apigenin, L-glutamic acid, 2-amino isobutyric acid, proanthocyanidin, and caffeic acid are weighed according to the mass ratio and mixed to obtain the premix; the premix and the Bletilla striata flower water extract are mixed to obtain the skin repair composition.

6. The production method according to claim 5, wherein The preparation method of the Bletilla striata flower water extract is as follows: Bletilla striata flower powder and water are mixed in a ratio of 1g:20mL-50mL, and extraction is carried out at 60-90℃ under reflux for 1-3h, and the filtrate is concentrated and freeze-dried to obtain the Bletilla striata flower water extract.

7. The skin repair composition of claim 1 for use in the preparation of a skin repair product.

8. Use according to claim 7, characterized in that, The skin repair product further comprises one or more than two combinations of a humectant, a thickening agent, an emulsifying agent, a chelating agent, a preservative, and a solvent.

9. Use according to claim 7, characterized in that, The solvent is water; The skin repair product is a solution with a concentration of 0.125mg / mL-2mg / mL prepared by diluting the skin repair composition with water.

10. Use according to claim 7, characterized in that, The repair is at least one of antioxidation, anti-aging, ultraviolet damage repair, and moisturizing. The repair is at least one of antioxidation, anti-aging, ultraviolet damage repair, and moisturizing.

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